Mitochondria were isolated from A. fumigatus mycelium (10 g moist mass) based on a previously described protocol (74 (link)). Wild-type and ΔfhdA conidia (1 × 108) were precultured on 100 ml of MM for 8 h at 37°C prior to addition of 2 μg/ml caspofungin and left to grown for 48 h to induce cell wall stress. The control was left untreated. Mycelia were harvested by filtration in Miracloth (Calbiochem), frozen in liquid nitrogen, and subjected to cell fractionation methods. Mitochondrial extracts were quantified by the use of a modified Lowry method (75 (link)) and loaded on 12% SDS-PAGE, electroblotted to PVDF (polyvinylidene difluoride) membranes (GE Health Care), and subsequently analyzed by Western blotting. Monoclonal VDAC1/porin antibody (16G9E6BC4) from S. cerevisiae (Thermo Scientific) and polyclonal homemade anti-CytC antibody (a gift from Mário H. Barros) were used to confirm mitochondrial membrane enrichment.
Pvdf polyvinylidene difluoride membranes
Manufactured by GE Healthcare
Sourced in China
PVDF (polyvinylidene difluoride) membranes are a type of laboratory equipment used for various applications in research and diagnostic settings. These membranes are made of a hydrophobic polymer material and are characterized by their chemical and thermal stability, as well as their ability to bind and immobilize a wide range of biomolecules, including proteins, nucleic acids, and other biological compounds. PVDF membranes are commonly used in techniques such as Western blotting, dot blotting, and immunoassays, where they serve as a support matrix for the separation and detection of target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Ai600 imager, by GE Healthcare (2 mentions)
Coomassie brilliant blue stain r 250, by Beyotime (2 mentions)
Chemidoc imager, by Bio-Rad (2 mentions)
Xt loading buffer, by Bio-Rad (2 mentions)
Nupage novex bis tris 4 12 midi gels, by Thermo Fisher Scientific (2 mentions)
Mops or mes, by Thermo Fisher Scientific (2 mentions)
Non fat dry milk protein, by Bio-Rad (2 mentions)
Non fat dry milk protein, by Roche (2 mentions)
Bicinchoninic acid (bca), by Thermo Fisher Scientific (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Enhanced chemiluminescence, by GE Healthcare (1 mentions)
G3pdh, by OriGene (1 mentions)
Ecl film, by GE Healthcare (1 mentions)
6 protocols using pvdf polyvinylidene difluoride membranes
1
Isolation and Characterization of Mitochondria from Aspergillus fumigatus
2
Quantitative Protein Analysis in Plants
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Total protein from plant samples was isolated using a phenol-based extraction method (Cahoon et al. 1992 ). Protein quality was determined using the Easy II Protein Quantitative Kit (BCA, TransGen Biotech, China). Samples of 25 µg of total fruit protein and a dilution series of total leaf protein were electrophoretically separated in 12% SDS-polyacrylamide gels. The protein in the gel was either stained with Coomassie Brilliant Blue R-250 stain (Biyuntian Biotechnology Co., Ltd., China) or blotted onto PVDF (polyvinylidene difluoride) membranes (GE Healthcare) using wet transfer for 1.5-2 h. The membranes were blocked in in TBS-T solution (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1% (v/v) Tween-20) supplemented with 5% (w/v) fat-free milk for 1 h at room temperature (RT), and subsequently incubated with primary antibodies against PsbA and PsbD (1:1000 dilution, PhytoAB) at 4 °C overnight.
Then the membranes were washed with TBS-T (10 min at RT, three times), stained with HRP conjugated anti-rabbit secondary antibody at 1/10000 dilution for 1 h at RT, and again washed with TBS-T (10 min at RT, three or more times). Detection was performed with the enhanced chemiluminescence (ECL, Biosharp, China) kit and the AI600 imager (GE Healthcare).
Then the membranes were washed with TBS-T (10 min at RT, three times), stained with HRP conjugated anti-rabbit secondary antibody at 1/10000 dilution for 1 h at RT, and again washed with TBS-T (10 min at RT, three or more times). Detection was performed with the enhanced chemiluminescence (ECL, Biosharp, China) kit and the AI600 imager (GE Healthcare).
3
Quantitative Protein Analysis in Plants
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Total protein from plant samples was isolated using a phenol-based extraction method (Cahoon et al. 1992 ). Protein quality was determined using the Easy II Protein Quantitative Kit (BCA, TransGen Biotech, China). Samples of 25 µg of total fruit protein and a dilution series of total leaf protein were electrophoretically separated in 12% SDS-polyacrylamide gels. The protein in the gel was either stained with Coomassie Brilliant Blue R-250 stain (Biyuntian Biotechnology Co., Ltd., China) or blotted onto PVDF (polyvinylidene difluoride) membranes (GE Healthcare) using wet transfer for 1.5-2 h. The membranes were blocked in in TBS-T solution (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1% (v/v) Tween-20) supplemented with 5% (w/v) fat-free milk for 1 h at room temperature (RT), and subsequently incubated with primary antibodies against PsbA and PsbD (1:1000 dilution, PhytoAB) at 4 °C overnight.
Then the membranes were washed with TBS-T (10 min at RT, three times), stained with HRP conjugated anti-rabbit secondary antibody at 1/10000 dilution for 1 h at RT, and again washed with TBS-T (10 min at RT, three or more times). Detection was performed with the enhanced chemiluminescence (ECL, Biosharp, China) kit and the AI600 imager (GE Healthcare).
Then the membranes were washed with TBS-T (10 min at RT, three times), stained with HRP conjugated anti-rabbit secondary antibody at 1/10000 dilution for 1 h at RT, and again washed with TBS-T (10 min at RT, three or more times). Detection was performed with the enhanced chemiluminescence (ECL, Biosharp, China) kit and the AI600 imager (GE Healthcare).
4
Western Blot Analysis of Protein Extracts
5
Quantifying Cellular Protein Levels
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We detected the amount of each protein in the cell lysate as previously described with slight modifications [2] (link). The cells were lysed with homogenization buffer as described above. After the protein concentration was determined, each cell lysate was dissolved with an equal amount of 2× sodium dodecyl sulfate (SDS)-Laemmli sample buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS and 10% glycerol). Equal amounts of cell lysate were separated on 10% SDS-polyacrylamide gels, immunoblotted onto polyvinylidene difluoride membranes (PVDF) (GE Healthcare) and identified by enhanced chemiluminescence (GE Healthcare) using antibodies against ATF4 (Santa Cruz Biotechnology), actin (Calbiochem) and G3PDH (Acris). To detect HiBiT-tagged protein on PVDF membranes, we incubated the protein-transferred membrane with TTBS for more than 15 min and soaked it with HiBiT reaction mixture containing rLgBiT and furimazine in diluted HiBiT lytic buffer for 5 min. The membrane was exposed to ECL film (GE Healthcare) for an appropriate time, and the images were obtained in the same manner as the immunoblot analysis.
6
Western Blot Analysis of Protein Extracts
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Brain cortical tissue, liver, mBCEC, or pBCEC were lysed by brief sonication in protein lysis buffer (containing 50 mM Tris, 10 mM EDTA, 1% Triton X 100, phosphatase inhibitor (PhosSTOP™), protease inhibitor cocktail (Roche Diagnostics cOmplete™), and pH adjusted to 7.5). Protein quantification was performed using bicinchoninic acid assay (BCA, ThermoFisher Scientific). Samples were mixed (4:1) with XT loading buffer (Bio-Rad) and proteins were denatured at 90 °C for 5 min. Proteins (20 μg/well) were loaded on NuPage® Novex 4–12% Bis-Tris Midi gels and separated under reducing conditions by SDS-PAGE using 1× MOPS or MES (Life Technologies) buffer. Then, proteins were electroblotted onto 0.45 μm polyvinylidene difluoride membranes (PVDF; GE Healthcare). Membranes were blocked with 5% non-fat dry milk protein (Bio-Rad) for 1 h at room temperature (RT). Membranes were incubated with primary antibody overnight in 5% bovine serum albumin (BSA; Roche) at 4 °C, followed by HRP-conjugated secondary antibody incubation in 5% non-fat dry milk protein for 1 h at RT. Detection of immune-reactive bands was performed using Clarity Western ECL Substrate (Biorad) and a ChemiDoc imager (Bio-Rad). Band intensities were determined using ImageLab software (version 5.2.1, Bio-Rad). All primary and secondary antibodies used are listed in Supplementary Table 1 .
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